DE3712334A1 - Process for the cultivation of HIV-2 viruses, cell cultures suitable for this purpose, and diagnostic agents for detecting HIV-2 viruses and process for their production - Google Patents

Abstract

The invention relates to a process for obtaining HIV-2 viruses in which a culture of permanent T-lymphocytes or of lymphocytes from cord blood is mixed with serum or lymphocytes of patients infected with HIV-2. This cocultivation is continued under suitable conditions. When cord blood lymphocytes are used, a mixing with permanently growing T-lymphoma cells is carried out after 14 days. After infection of the infectable cells there is an adequate measurable virus concentration in the culture supernatant so that the viruses can be isolated and investigated in a conventional way. Also described is a process for detecting HIV-2 antibodies in samples, in particular blood samples, in which a cell-free sample is subjected to an antigen-antibody reaction with a viral antigen obtained by the abovementioned process, and the extent of this reaction is determined in a conventional way, as well as agents for carrying out this process which contain an appropriate HIV-2 virus preparation as well as reagents for detecting HIV-2 virus/HIV-2 antibody complexes.

Description

The invention relates to a method for growing HIV-2 viruses, suitable cell cultures as well as diagnostic ones Means for the detection of HIV-2 viruses and methods for their Manufacturing.

AIDS (Acquired Immune Deficiency Syndrome), a self worldwide epidemic spreading disease is caused by HIV viruses (Human Immunodeficiency Virus) - also HTLV III (Human T-cell leukemia virus type III) or LAV (Lymphadenopathy Associated Virus) - caused (Science 220, 859 (1983). Detection of this disease, the can have a long latency period Detection of HIV antibodies found in the patient's blood Find. There are several commercial tests of this type now known and routinely used to on the one hand to diagnose AIDS diseases and on the other hand check blood supplies to prevent spread to prevent the disease from spreading blood.

Recently it has now been found that in addition to the known HIV viruses (hereinafter referred to as HIV-1), and others AIDS-causing viruses occur in certain patients and with the known HIV-1 tests not or only weakly react. These are referred to below as HIV-2. There the risk of infection and illness with HIV-2 as well As serious as with HIV-1, it was diagnosed and diagnosed Prophylaxis necessary, also suitable diagnostics against HIV-2 To find viruses.

The invention is therefore based on the object and To provide procedures that allow HIV-2 viruses manufactured and HIV-2 antibodies quickly and safely in corresponding samples can be detected.  

This task is characterized by the in the claims measures resolved.

• A. Extraction of HIV-2 Viruses
  • 1. Patient selection
    Sera from risk patients for HIV infection were tested for HIV-1 antibodies in a Western blot, with immunofluorescence and in various commercial ELISAs. Sera that reacted only weakly in the test systems mentioned were then tested for HIV-2 (HTLV IV) antibodies in a Western blot. In this way, patients could be selected who had little or no reaction in the HIV-1 test, but who reacted significantly in the HIV-2 test.
  • 2. HIV-2 cultivation
    The cultivation of HIV-2 viruses can be done by directly infecting a suitable T lymphocyte culture (e.g. HUT 78, Molt 3, K 37, CEM or HT, as described in WO 85-04 897) with the serum HIV-2 positive patients. For this purpose, such a culture, which contains approximately 10 ⁶ to 10 ⁷ cells / ml in a suitable culture medium and is mixed with approximately 2 μg / ml polybrene, is mixed with approximately 10% of the infected serum and under a CO ₂ atmosphere at 37 ° C. held (1-6 weeks) until syncytia formation and blistered degeneration of the cells indicate virus production.
  • The viruses can be obtained in a known manner from this culture, for example by adding sodium chloride / polyethylene glycol to the supernatant and centrifuging off the precipitate at about 2000 rpm or by ultracentrifugation.
  • Further cleaning can e.g. B. by centrifugation in density gradient, the HIV-2 viruses, like other retroviruses have a density of 1.16.
  • However, it is more advantageous than infection with serum to separate the lymphocytes from the blood of patients infected with HIV-2 and this in a suitable culture medium which contains phytohemagglutinin (PHA) and interleukin-2 (TCGF-T-cell growth factor) for growth stimulation ent contains to grow for about 3 days and then to pass 1-3 times on a cell culture which reacts to HIV cythopathogen, for example umbilical cord lymphocytes, before this culture in a dilution of 1:10 to 1: 5 to infect the above permanent cells -Lines is used. The rapidly occurring cytopathogenic effect allows cultures that are not infected with HIV-2 to be quickly recognized and sorted out.
  • As soon as a corresponding HIV-2 cell culture is obtained, it will be used for further infections and the detour through patient selection will be saved.
  • The HIV-2 viruses used below are deposited as culture in HUT 78 cells in accordance with Art. 28 Budapest Treaty with the European Collection of Animal Cell Cultures (PHLS), Porton Down, Salisbury, England under number HIY-2 / PEI 402.
• B. Detection of HIV-2 antibodies
  The detection of HIV-2 antibodies is based in each case on the specific antibody / antigen reaction with the HIV-2 viruses and the detection of the complexes formed. In addition to the virus itself, an active fragment, ie a special protein from the envelope or the core of the virus, or virus-containing cells can also be used as the antigen.
  • 1. Western blot test
    The basics of this method are from Towbin, Proc. Nat. Acad. Sci, USA 76, 4350-4354 (1979). Analogously to this, the HIV-2 viruses are lysed, the proteins contained are separated electrophoretically on sodium dodecyl sulfate (SDS) -containing polyacrylamide gel and transferred electrophoretically to a nitrocellulose layer. Then protein-binding sites present on the nitrocellulose are saturated with serum albumin, the layer is dried and cut into strips parallel to the direction of electrophoresis. When incubated with a test serum, only the HIV-2 antibodies that can react with the HIV-2 virus proteins are now bound and other proteins contained in the serum are washed out. When incubated with a labeled anti-human IgG or IgM antiserum, this then in turn attaches itself to the HIV-2 antibody, so that the ternary complex can now be detected on the basis of the labeling. As a label, it is common to substitute the tyrosine components of the antibody protein yourself with 125-iodine and to measure the radioactivity of the complexes thus formed by placing a photo paper on the nitro cellulose strip (RIA method) or the antibodies with an easily detectable enzyme ( To couple peroxidase; alkaline phosphatase galactoseidase) and to stain the complex by reacting with the appropriate reagents (e.g. H ₂ O ₂ / benzidine derivative; p-nitrophenyl phosphate; galactoside / leuco dye) (ELISA method) or the antibody to a fluorescent dye bind and evaluate the fluorescence of the complex formed directly (IFA method).
  • 2. ELISA method (enzyme linked immunoadsorbent assay)
    For this test, the wells of a microtiter plate made of polystyrene are coated with about 0.5 μg protein lysate from HIV-2 viruses or a corresponding amount of the viruses in a buffer solution and fixed by drying or precipitation with alcohol or acetone. Additional binding sites for protein are saturated with bovine serum albumin before the test serum is added, so that only HIV-2 protein antibodies are bound to the corresponding antigens. A detectable ternary immune complex is then formed by reaction with an enzyme-labeled anti-human IgG or IgM antiserum (see above). By binding the antigen to the carrier surface, it is possible to wash out excess reagent between the individual incubation steps and thereby avoid disturbances of the next reaction. The color reaction of the ternary complex is stopped after a defined time and the result is usually evaluated with a scanner.
  • Instead of the microtiter plates, it is also possible to bind the virus antigens to the wall of a cuvette or to beads made of polystyrene and thus to modify the test arrangement in a manner known per se.
  • Instead of a polystyrene surface, which is known to fix proteins, other suitable materials can also be used.
  • Furthermore, it is possible, instead of the simple "sandwich" method described, to fix the label over several "intermediate antibodies" or to add a defined amount of a labeled HIV-2 antigen to the test serum and thus to form a "competitive" test. For further variations, reference is made to the numerous publications of these methods.
• 3. IFA (Immunofluorescence assy)
  In this test, cells containing HIV-2 viruses are placed directly on a slide in a buffer solution (approx. 50 µl), dried and fixed with alcohol / acetone. The tests can be stored at -20 ° C in this state. To carry out the test, approx. 20 µl test serum 1:10 in PBS (phosphate buffered saline) is added and incubated for 1 hour at 37 ° C., washed and stained with a dilute solution of fluorescein-conjugated anti-human IgG or IgM and the fluorescence reduced evaluated under a microscope.
  • The above methods are only intended to provide general insight. Methods known to the person skilled in the art for cell cultivation and multiplication of viruses as well as methods known for other immunoassays and to be used accordingly should therefore also be encompassed by the invention.
  • With simultaneous use of HIV-2 and known HIV-1 virus preparations according to the invention, suitable means for the detection of both viruses can be produced, in particular for the examination of preserved blood.
  • In the following examples, preferred configurations are given without the invention being restricted thereto.

example 1 Production of HIV-2 viruses

From 10 ml of heparinized whole blood from an HIV-2 positive patient the mononuclear cells were ge over a Ficoll gradient cleans. The purified mononuclear cells were used for 3 days PHA stimulated and with 10% Interleukin 2 RPMI 1640 (20% FCS) cultured. There was a three-time passage on the navel cord lymphocytes that have been enriched in the above-mentioned manner. Then there was a cell-bound transfer to the permanent T-lymphoma line HUT 78. These cultures were permanently treated with 2 µg / ml Polybrene kept. After about 4 weeks of cell cultivation there was a typical cytopathic effect for the HIV group with syncytia formation and subsequent blistering degeneration of the cell len. Evidence of the virus production now beginning was over performed a reverse transcriptase detection.

For this test, 30 ml of cell culture supernatant at 100,000 G Centrifuged for one hour and then the pellet in the RT assay tested. The RT test gave 40,000 CPM with a background from 2000 CPM.

The Western blot, carried out in the manner described above obtained antigen, gave the typical band pattern of HIV-2.

Example 2 Immunofluorescence test

HIV-2 infected and uninfected T lymphocytes (HUT 78) are washed twice in PBS and resuspended in PBS at 4-6 × 10 ⁷ cells / ml. 2 µl (approx. 10 ⁵ cells) of the infected and non-infected cells are applied separately to masked, degreased slides and dried overnight at room temperature.

One pair of slides each with 20 µl test and con troll serum in a dilution in PBS of 1:20 and 1:40 be opened and incubated at 37 ° C for 30 min in a humid chamber. Then it is washed twice with PBS for 10 minutes, dried, with 20 μl FITC-labeled anti-human IgG applied, again for 30 min Incubated at 37 ° C and washed. The cells stained this way Preserved with 50% glycerin in PBS and with a fluorine escence microscope (Leitz HBO 200; 300x magnification) rated.

Example 3 ELISA test

5 mg / ml of highly purified HIV-2 viruses are mixed 1: 1 with a solution of 1% Triton X-100 in 5 mM sodium carbonate buffer pH 9.6 and heated at 56 ° C. for 30 minutes in order to inactivate the viruses. The suspension is cooled to room temperature, diluted 1: 500 with 50 mM sodium carbonate buffer pH 9.6, filled into ELISA plastic wells and kept at 4 ° C. for 16 hours in order to bind the antigens to the vessel wall. The rest of the solution is then poured out and the cups are washed with distilled water and dried. Now the wells are filled with 200 ul of a solution of 0.1% rinder serum albumin and 2% rabbit serum in 50 mM sodium carbonate buffer pH 9.6, incubated for 1 hour at 37 ° C. and with 0.05% Tween-20 in PBS washed before 100 μl of test solution (serum 1: 100 diluted with PBS + 005% Tween) is poured in and incubated at 37 ° C. for 90 min. Residues of the test serum are removed by washing twice with 0.05% Tween-20 and 0.5% rabbit serum in PBS and washing twice in 0.5% rabbit serum in PBS. Then 100 .mu.l of a solution of rabbit anti-hum IgG, conjugated with peroxidase 1: 1000 in 0.05% Tween-20 in PBS is added, incubated for 60 min at 37 ° C and washed 4 times as before. A solution of 10 mg of o-phenylenediamine and 10 μl of H ₂ O ₂ (30% in phosphate buffer pH 6.8) is then added, the mixture is kept at 20 ° C. for 30 minutes and the reaction is stopped with 2 N sulfuric acid. The reaction is measured using the yellow color at 486 nm.

Example 4 Western blot test

150 mg HIV-2 viruses are in a solution of 350 ul buffer (2% SDS, 2% glycerol, 0.1 M Tris-HCl pH 6.8, 20 mM PMSF (phenylmethylsulfonyl fluoride), 0.2% bromophenol blue, 0, 5% β- mercaptoethanol) lysed, applied to a 16 cm × 16 cm SDS polyacrylamide gel plate and the virus proteins were electrophoresed using a voltage of 180 V. After a time of approx. 4 h, a nitrocellulose filter is placed on top and the protein bands are transferred electrophoretically with a voltage of 60 V in 3.5 min. The nitrocellulose filter is soaked with 1% BSA (bovine serum albumin) and 1% gelatin in PBS, pH 7.4, dried and cut into strips 0.5 cm wide. For use, the strips are moistened with a buffer solution 1 consisting of 10% fetal calf serum, 1% BSA and 0.1% Triton X-100 in PBS for 90 min at 20 ° C and then 1 strip each with a 1: 100 dilution of the test serum in incubated above buffer 1. The incubation time depends on the temperature. 90 min at 37 ° C, 120 min at 20 ° C and 16 hours at 4 ° C have proven their worth.

The strips are after incubation 3 times 15 min with the Puf fer 1 washed before with a 1: 1000 dilution of anti-human IgG - anti-serum, peroxidase - conjugated in 2.5 ml of buffer solution 1 for 60 min at 20 ° C were .

Excess antiserum is 3 times 15 min with buffer solution 1 and Washed 3 times for 15 min with PBS, pH 7.4.

The strips are stained with a solution of 50 mg 3.3- Diaminobenzidine and 50 ul hydrogen peroxide in 100 ml PBS, pH 7.4 Incubated for 20 min at 20 ° C. The reaction is carried out by 3 times 15 min Wash stopped with distilled water, the strips dried and evaluated photometrically. It shows up with the HIV-2 Antibody-positive sera are the bands characteristic of HIV-2 at 26 kd, 32 kd, 51 kd, 55 kd, 65 kd, 130 kd. With the cross test with Known HIV-1 positive sera result in no, or only one weak reaction against p 26 (gag) and / or yp 32 (env).  

All three of the above tests can also be used in the same way a mixture of HIV-1 and HIV-2 preparations with a simultaneous test for both antibodies. Of course, only in the Western blot test by different bands differentiate.

Claims (11)

1. A method for obtaining HIV-2 viruses, characterized in that a culture of permanent T-lymphocyte cells with serum or leucocytes from an HIV-2 infected patient or with an HIV-2 infected cell culture and cultivated under suitable culture conditions and after the formation of a sufficient virus concentration from the culture, the viruses or virus-containing cells are isolated in the usual way. 2. The method according to claim 1, characterized in that the im Serum or contained in leucocytes of infected patients HIV-2 viruses first one or more times in lymphocyte Cultures are passed on and enriched on them have a strong cytopathic effect before they become permanent Cell culture are transferred. 3. The method according to claim 1 or 2, characterized in that the permanent cell culture is HUT 78, Molt 3, CEM or HT. 4. Permanent T lymphocyte cell culture, which ent HIV-2 viruses holds. 5. Cell culture according to claim 4, characterized in that it is a HUT 78 culture known as HIV-2 PEI 402 is deposited with PHLS. 6. Method for the detection of HIV-2 antibodies in samples, ins special blood tests, characterized in that the cell-free sample with one according to any one of claims 1-3 antigen-antibody reaction lets go and the extent of this reaction in itself determined in a known manner. 7. The method according to claim 6, characterized in that as Test method a Western blot, ELISA, or immunofluores zenzverfahren is applied.   8. The method according to claim 6 or 7, characterized in that for the simultaneous detection of HIV-1 and HIV-2 antibodies the sample with a preparation containing HIV-1 and HIV-2 viruses contains is reacted. 9. Containing agents for the detection of HIV-2 antibodies HIV-2 virus preparations according to any one of claims 1-3 and HIV-2 Virus / HIV-2 Antibody Detection Reagents Complexes. 10. Composition according to claim 9 for use in a western Blot, ELISA, or immunofluorescence methods. 11. A composition according to claim 9 or 10, characterized in that for the simultaneous detection of HIV-1 antibodies an HIV-1 Virus is included.